Virus Evolution, 2021, 7(1): veab032

doi: 10.1093/ve/veab032
Research Article

Downloaded from https://academic.oup.com/ve/article/7/1/veab032/6206434 by Universidade Federal de Viçosa user on 25 March 2022

Interannual dynamics, diversity and evolution of the
virome in Sclerotinia sclerotiorum from a single crop field
Jichun Jia,1,2 Yanping Fu,2 Daohong Jiang,1,2 Fan Mu,1,2 Jiasen Cheng,1,2
Yang Lin,2 Bo Li,1,2 Shin-Yi Lee Marzano,3 and Jiatao Xie1,2,*,†
1
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070 Hubei
Province, People’s Republic of China, 2Hubei Key Laboratory of Plant Pathology, College of Plant Science and
Technology, Huazhong Agricultural University, Hubei Province, Wuhan 430070, People’s Republic of China
and 3United States Department of Agriculture/Agricultural Research Service, Toledo, OH 43606, USA.
*Corresponding author: E-mail: jiataoxie@mail.hzau.edu.cn
†
https://orcid.org/0000-0003-1961-0338

Abstract
Mycovirus diversity is generally analyzed from isolates of fungal culture isolates at a single point in time as a snapshot. The
stability of mycovirus composition within the same geographical location over time remains unclear. Not knowing how the
population fluctuates in the field can be a source of unpredictability in the successful application of virocontrol. To better
understand the changes over time, we monitored the interannual dynamics and abundance of mycoviruses infecting
Sclerotinia sclerotiorum at a rapeseed-growing field for three years. We found that the virome in S. sclerotiorum harbors unique
mycovirus compositions each year. In total, sixty-eight mycoviruses were identified, among which twenty-four were
detected in all three successive years. These twenty-four mycoviruses can be classified as the members of the core virome
in this S. sclerotiorum population, which show persistence and relatively high transmissibility under field conditions. Nearly
two-thirds of the mycoviruses have positive-sense, single-stranded RNA genomes and were found consistently across all
three years. Moreover, twenty-eight mycoviruses are newly described, including four novel, multi-segmented narnaviruses,
and four unique bunyaviruses. Overall, the newly discovered mycoviruses in this study belong to as many as twenty
families, into which eight were first identified in S. sclerotiorum, demonstrating evolutionarily diverse viromes. Our findings
not only shed light on the annual variation of mycovirus diversity but also provide important virus evolutionary clues.

Key words: Sclerotinia sclerotiorum; core mycoviruses; interannual dynamics; diversity; evolution.

1. Introduction friendly biological controls, including virocontrol with hypovir-

Many fungi are pathogenic and cause severe disease in a variety
of plants and animals, including humans (Sharon and
Shlezinger 2013). Phytopathogenic fungi are responsible for ap-
proximately 70-80% of plant diseases (Oerke 2006). When resis-
tant cultivars are not available, the application of chemical
fungicides is the primary method for controlling fungal diseases
in economically important crops. However, environmentally
ulence-associated mycoviruses, have been considered a viable
alternative approach (Garcı́a-Pedrajas et al. 2019).
Viruses are the most abundant and the most diverse
organisms that exist almost everywhere on Earth (Güemes et al.
2016). They infect all types of life forms, from animals and
plants to microorganisms, including bacteria and fungi (Koonin
et al. 2006). Metagenomics or metatranscriptomics have

C The Author(s) 2021. Published by Oxford University Press.

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1
 2 | Virus Evolution, 2021, Vol. 7, No. 1
revealed a remarkable diversity of viruses sampled from various
cellular organisms in the past few decades (Zhang et al. 2019).
The uncovering of the virosphere diversity has rapidly trans-
formed our understanding of virus diversity and evolution
(Zhang et al. 2018). A typical virus has a coat protein that encap-
sidate the viral genome to form a virion (Krupovic and Koonin
2017). However, many positive-sense single-stranded RNA vi-
ruses are capsidless, including Narnaviridae, Mitoviridae,
Hypoviridae, Endornaviridae, Umbravirus, among several others
(Dolja et al. 2012).
Mycoviruses replicate in all lineages within kingdom Fungi
and have been described in phytopathogenic, entomopatho-
genic, medical, and edible fungi (Pearson et al. 2009; Xie and
Jiang 2014; Ghabrial et al. 2015; Kotta-Loizou and Coutts 2017).
Most mycovirus infections cause few or negligible symptoms
detectable in the host (Ghabrial and Suzuki 2009). However, a
portion of the mycoviruses can reduce fungal growth, sporula-
tion, virulence, and mycotoxin production and cause fruiting
bodies to deform (Xie and Jiang 2014). Mycoviruses have also
been reported to enhance abiotic stress tolerance, pathogenic-
ity, or the fitness of their fungal host (Ahn and Lee 2001;
Marquez et al. 2007; Ozkan and Coutts 2015). In practical use,
some of the mycoviruses known to induce hypovirulence have
been developed as virocontrol agents against plant fungal dis-
eases (Nuss 2005; Ghabrial et al. 2015).
Sclerotinia sclerotiorum is a devastating phytopathogenic fun-
gus with a worldwide distribution and can infect more than 700
species of plants, including important crops and numerous
weeds (Farr and Rossman 2020). Sclerotinia stem rot caused by
S. sclerotiorum is one of the most serious diseases of annual
rapeseed, potentially resulting in yield losses as great as 80%
and significantly reducing oil content and quality (Sharma et al.
2015). Sclerotinia sclerotiorum harbors a great diversity with
eighty-nine mycoviruses documented in the GenBank database
(update date: 26 September 2020), some of which have great po-
tential to be developed as virocontrol agents. For instance, a cir-
cular single-stranded DNA virus that confers hypovirulence has
been shown to manage sclerotinia disease effectively under
field conditions (Yu et al. 2010; Yu et al. 2013; Zhang et al. 2020).
In this study, to investigate the spread and prevalence of
mycoviruses in S. sclerotiorum, we focused on mycovirus biodi-
versity and the annual variation within a single field for three
years at the end of growing seasons. The results suggest that
even within a small geographical area, the S. sclerotiorum virome
consists of a highly variable mycovirus community that not
only includes a stably maintained core population under field
conditions but also harbors a dynamic portion of the mycovirus
compositions. We aimed to define novel mycoviruses in explor-
ing the diversity and evolution of viruses detected from a field
population of S. sclerotiorum, as well as initiating a platform for
continuously monitoring fungus–mycovirus dynamics in natu-
ral fungal communities.
2. Materials and methods
2.1 Fungus isolation and purification
From the pith of rapeseed stems in the late stage of the disease,
sclerotia were collected annually for three years from 2016 to
2018. More than 100 sclerotia were collected each year from dis-
eased stems (one sclerotium from each diseased stem) that
were randomly sampled from a single field (approximately
667 m2) located in Wuxue City (N29 590 3.3600 , E115 270 58.8400 ),
Hubei Province, China. All sclerotia were isolated and purified
to obtain 100 isolates of S. sclerotiorum. A total of 300 isolates
were purified for three years. All isolates were cultured on po-
tato dextrose agar at 20  C and stored at 4  C.
2.2 Total RNA extraction and RNA sequencing
Total RNA of each isolate was extracted from mycelia using an
RNAiso Plus kit (TaKaRa, China) according to the manufac-
turer’s instructions and then treated with DNase I. The concen-
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tration of each RNA extraction was measured using a Nanodrop
2000 spectrophotometer (Thermo Fisher Scientific, USA). Total
RNA was stored at 80  C until being used for RNA-Seq analysis.
Equal amounts of total RNA from 100 isolates collected annually
were pooled in a single library according to the collection year.
Three RNA libraries were generated and used for sequence
analysis. All library preparations and sequencing steps were
performed by GENEWIZ Inc. (China). rRNA was depleted from
total RNA using the Illumina Ribo Zero rRNA removal Kit (Plant
Leaf). Three libraries were constructed according to the manu-
facturer’s protocol (NEBNext Ultra Directional RNA Library Prep
Kit for Illumina). Paired-end (150 bp) sequencing of the three
cDNA libraries was performed on the HiSeq 2500 platform
(Illumina, USA). To obtain the 5’ and 3’ terminal sequences of
the viral genome, the previously described method was used
(Potgieter et al. 2009). Primers used in this study are listed in
Supplementary Table S1.
2.3 Virus-related sequence assembly and annotation
Adapter sequences and low-quality reads were removed from
the raw reads of each library using the Trimmomatic program
(version 0.36) (Bolger et al. 2014) with default parameter set-
tings. The remaining reads were mapped to the S. sclerotiorum
genome (http://fungi.ensembl.org/Sclerotinia_sclerotiorum_
1980_uf_70_gca_001857865/Info/Index) using HISAT2 (version
2.1.0) (Kim et al. 2015). Unmapped reads were extracted from
the BAM file using SAMtools (version: 1.9) (Li et al. 2009) and
were then assembled de novo using Trinity (version 2.5.0) (Haas
et al. 2013) and SPAdes (version 3.11.1) (Bankevich et al. 2012).
After the removal of all contigs shorter than 1,000 bp, the
remaining contigs were filtered for redundancy at 90% nucleo-
tide identity using CD-HIT (Fu et al. 2012). To identify virus-re-
lated contigs, the assembled contigs were compared against the
NCBI nonredundant protein database using DIAMOND BLASTX
(version 0.9.10) (Buchfink et al. 2015). We set the e-value below
1  105 to maintain high sensitivity and a low false-positive
rate. Contigs matched to the same reference sequence were
merged into longer contigs using DNAMAN software (version
8.0; Lynnon Biosoft, Quebec, Canada). Nucleotide sequences, po-
tential ORFs, and deduced amino acid sequences were analyzed
using DNAMAN.
2.4 Quantification of relative transcript abundances
We used the assembled contigs and S. sclerotiorum transcripts as
templates for mapping with the program Bowtie2 (Langmead
and Salzberg 2012). Next, we measured the abundance of these
sequences as the number of transcripts per million (TPM) within
each sequencing library. The relative abundance of each viral
transcript was calculated as the TPM using RSEM (Li et al. 2011)
and visualized in a heat map by TBtools (Chen et al. 2020).

2.5 Phylogenetic analysis of mycoviruses
To determine the evolutionary relationships among the newly
identified viruses, the RdRp encoded by the putative mycovi-
ruses was subjected to BLASTp analysis with the NCBI-nr data-
base. The core conserved domain of RdRp was aligned with
MAFFT (version 7.427) using the E-INS-i settings (Nakamura
et al. 2018). All alignments were trimmed with trimAl (v1.4)
(Capella-Gutierrez et al. 2009) to remove low-quality regions
through heuristic selection under the automatic method (-auto-
mated1) based on similarity statistics. The maximum likelihood
(ML) phylogenetic trees were constructed using the IQ-TREE
(version 1.6.11) (Nguyen et al. 2015) with 1,000 bootstrap repli-
cates and the best-fit amino acid substitution models were
identified using ModelFinder (Kalyaanamoorthy et al. 2017).
Nodes supported by a bootstrap value below 70% were collapsed
in TreeGraph 2 (Stöver and Müller 2010). Phylogenetic trees
were visualized in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/soft
ware/figtree/). The percent identity matrix among protein-cod-
ing sequences was generated using Clustal Omega for multiple
sequence alignment (Li et al. 2015).
2.6 Availability of data and materials
The raw sequence reads from the metagenomic libraries are
available at the NCBI Sequence Read Archive (SRA) database un-
der BioProject accession number PRJNA598316. The sequences
reported in this research have been deposited in GenBank data-
bases, and the assembled contig sequences are available from
GenBank databases under the accession numbers indicated in
Supplementary Table S2. All virus nucleotide sequences (Fasta
format), the unaligned and aligned datasets used in the phylo-
genetic analyses (Fasta format), and the phylogenetic trees
(Newick format) that were generated in this work are available
at the Figshare website (DOI: 10.6084/m9.figshare.11474709).
3. Results
3.1 Metatranscriptomic identification of mycoviruses
from S. sclerotiorum in a single rapeseed field
We initially analyzed the growth rate, colony morphology, and
pathogenicity for the 100 isolates of S. sclerotiorum collected in
May 2017, and the results showed that these isolates were sig-
nificantly different in these three biological characteristics
(Supplementary Fig. S1). Nearly 50% of the isolates grew fast
and had strong pathogenicity, while some isolates grew slowly
with reduced pathogenicity (Supplementary Fig. S1C and D).
These phenotypic differences indicate that there may be abun-
dant mycoviruses in the selected field site. To understand the
interannual dynamics in diversity and abundance of mycovi-
ruses in this field, a total of 300 isolates were collected and used
to construct three metatranscriptomic sequencing libraries
according to the collection year. Total coverage of more than
100 million reads passing the filtering criteria was obtained for
each library. For simplicity, we named the three libraries
2016SS, 2017SS, and 2018SS based on the collection year. Based
on the analysis of the assembled contigs, a total of sixty-eight
putative mycoviruses with nearly complete genomes (contain-
ing complete ORFs) or partial genomes were identified
(Supplementary Table S2).
J. Jia et al. | 3
3.2 Novel positive-sense single-stranded RNA viruses
In this study, the forty-three putative viruses encoded by fifty-
two contigs as diverse as previously characterized þssRNA vi-
ruses (Supplementary Table S2), signifying that S. sclerotiorum
does harbor abundant þssRNA mycoviruses with great diversity
regardless of the geographical origins.
Four novel multi-segmented narnaviruses were identified,
which were temporarily named as Sclerotinia sclerotiorum nar-
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navirus 1 (SsNV1), SsNV2, SsNV3, and SsNV4, representing the
first discovery of narnaviruses in S. sclerotiorum. RT-PCR analysis
of the cDNA of each isolate included in the NGS sample 2017SS
showed that all segments of the four segmented narna-like vi-
ruses coexisted in the corresponding fungal isolate
(Supplementary Fig. S2). The 5’ and 3’ terminal sequences of the
four multi-segmented narnaviruses were obtained. The 5’ and
3’-terminal 40 nt were highly conserved in the multi-segments
of each narnavirus (Supplementary Fig. S3A). SsNV1 and SsNV2,
respectively, have quad- and tri-segmented genomes (Fig. 1A).
Interestingly, complete RdRps of SsNV1 and SsNV2 were
encoded by two different RNA segments. RNA1 of SsNV1 and
SsNV2 harbored the G, F, A, and B motifs of the RdRp at the C-
terminus, whereas RNA2 of these two viruses contained the C,
D, and E motifs at its N-terminus (Fig. 1A and Supplementary
Fig. S3B). RNA1 of SsNV1 and SsNV2 encoded proteins share less
than 62% identity to other narnaviruses, while proteins encoded
by RNA2, RNA3, and RNA4 of these two viruses share no signifi-
cant similarity with known sequences in either the NCBI nr
database or in the Pfam domain database. In addition, the other
seventeen narnaviruses with multi-segmented genomes were
identified from public data (NCBI SRA and TSA database) (Fig. 1B
and Supplementary Table S3). The phylogenetic tree suggested
that these divided RdRp narnaviruses formed an independent
phylogenetic cluster, which was significantly distant from other
non-segmented narnaviruses (Fig. 1B). Therefore, we propose a
new family, Polynarnaviridae, to accommodate this virus clade.
SsNV3 and SsNV4 have bi-segmented genomes (Fig. 1A). The
RNA1 segment contains an ORF encoding an essential RdRp,
and the RNA2 fragment contains an ORF encoding no similarity
with the known proteins in the NCBI nr database. BLASTp
analysis showed that the protein with the top hit for ORF1 of
SsNV3 was RdRp from Plasmopara viticola lesion-associated
orfanplasmovirus 4 (PvLaOrfPlV4) (33.75% identity), whereas
that for ORF1 of SsNV4 was RdRp from PvLaOrfPlV5 (72.16%
identity) (Supplementary Table S2). However, different from the
genomes of PvLaOrfPlV (1–5) that were reported to contain a sin-
gle RNA segment, SsNV3 and SsNV4 have two RNA segments.
Interestingly, the proteins encoded by RNA2 of SsNV3 are most
similar to coat proteins of the known viruses, based on the pre-
diction by I-TASSER and Phyre2 (Supplementary Fig. S4).
Therefore, we speculate that the genomes of SsNV3 and SsNV4
may not be naked, but encapsulated in virions. Phylogenetic
analysis demonstrated that SsNV3 and SsNV4 grouped as a
cluster with those unsegmented narnaviruses, but clustered
into a separate branch with PvLaOrfPlV (1–5) (Fig. 1B).
Twelve novel mycoviruses share RdRp identity with mem-
bers of the Botourmiaviridae family, ranging from 23% to 82%
(Supplementary Fig. S5A). The obtained genomes ranged from
1.99 to 3.85 kb in length, and each genome contained a single
complete ORF (Supplementary Fig. S5B). Phylogenetic analysis
suggested that twelve botourmiaviruses clustered into three
well-supported clades, among which five botourmiaviruses be-
long to the genus Penoulivirus, four belong to the genus
 4 | Virus Evolution, 2021, Vol. 7, No. 1

2568 1 3453
A RNA1 5' G F A B 3'
RNA1 5' RdRp 3'
42 2513

SsNV3
2285 8 3271
RNA2 5' C D E 3' 1 3100
SsNV1
50 2188
173 523 664 1020 RNA2 5' Putative coat protein 3'
8 2874
1168
RNA3 5' 3' 1 3105
42 1088
257 505 RNA1 5' RdRp 3'

SsNV4
7 2970
899
RNA4 5' 3' 1 2477

Downloaded from https://academic.oup.com/ve/article/7/1/veab032/6206434 by Universidade Federal de Viçosa user on 25 March 2022

43 768
RNA2 5' Putative coat protein 3'
55 318
2384 7 2358
RNA1 5' G F A B 3'
38 2302
SsNV2

2259
RNA2 5' C D E 3'
50 2173
730 1026
Nar
nav

PvNlV1 10
PVaNarna14
PVaNarna13
PVaNarna 30

PVaNarna9
PVaNa a11
3'

PVaN
RNA3 5' irid

PVa eyNLV1
PVaN

PVaNarna

PVa Narna 25
PVa arna38
1099

PvL aOrfP 4
ae
PVa arna3 7
56 805

BNlV22

Pv aOrfP lV2
PVa arna 4

BNlV23

PvL Narna2
WiNV2
NpNV1

Leps V1

Pv LaOrf lV1
PVa Narna 35

PiRV-4
PV

Pv LaOr PlV3
Narn

arn
N

PserN
PV Narn na36

arna3 2

Ss NV4 rfPlV 4
rna
N

Ss LaO fPlV
PV aNar NV1 0

5
a
N
PV Gn a2 1

aN

26
a3
a
arn 12
PV Hp SP a7

S N 20 a1
3

B
1
aN afN V1

a
3

RmcNV arn
S aN 3
a

P NlV V- S
a

FO NV

Sc VaN 4 20S
arn V
PV
Om ar V

S 21
aN PvN V2 1

2 3 rna
NV a
S
100
n 1
Af faN

PV 23 40
M
uN V

H
aN laN V- na
N ar 1
100
100
Af arn V3 m N V
e

R Va nN 1
100
ida

uN a6
100

P we NV
10

F
10

100 1 0 0 0
PV N pNV V1

10
0

B lun V2
0
vir

100

aN pN 2
100

100 B NL 1
O vNV 1

0
ar V 2
95
rna

10
PV
10
100

n
aN RsN a5 C xNV 1

0
1
0
10

C NLV rna4

10
arn V1
0
yna

100
95

PV MfaN a19 O aNa V1

10
0

100

P V uN L n a 4 2
10

a 10 1 0
PV Narn V3
0

10 0
Pol

0 0
Lh a N a r a 1 5
100

aN
a rn a 3
0
10
PV aNarn a43
10
97

0 10
AtN a 4 10 0
74

0
PV aNarn 23
10

0 10

10
97

Hla V 0
0

NV21
80
PV Narna 2
98

1 00

H
P V a N a rn a 2
3
PVa laNV1
10

0 99 9

0
0

99 0 10
Narn PVa Narn a 18
96

95 10
0
0
10
100

10

PVaN a2 10 10
1 100 PVa arna17
0 10

arn a 10000
0
10
0
SsNV 1 PVaN 1
10

1 00
99

10 0
Mm a N 1 100 99
10
0
10 0 FpNV rna16
V
MmaNV 3
100
1 74
PVaNa
10 0
SsOV5
10 0
99

MmaNV2
100 77
SsNV2 70 10 0 99
100
100 SsOV4
100 PVaOurmia91

Penoulivirus
CtNV1 100 99 100
76
HpafNV2 100
PmOLV1
98 80 84 CuOulV2
MfaNV2 100
100 84 SsOV7
100
100 SsOV6
100 100 AnOlV1
10 0

0
92 PlRV1
10 0 10 10
94 0 PoOLV
idae 98 7 79 100 100 SsOV9
Levivir
100 79 6
96 100 99 93
10
0 10
0 SsOV
98 10 10 10 74 SsOV 8

Boto
1 00 99 0 10 0
0
B631 SsO 13
85

10
V1-N
0 0
73
10

SsO V12
0

1
CpM DapiMV 1
10

99
0

SsO V3

Boto
rMV
10

80

u
100

Ama uMV1
0

EnO V2
0

r
10
10

99
92

mia
85

Bev aMV1
10 0

BO ulV1
10
10

100
0

uliv
Ss LV
0

Cas qMV17
96
100

10
100

Ss OV11
vir
0
0

C V2
10
99
10

100

M 9 Ss OV1
92

iru
100

Ss MV2 8
100
0

99

ida
0

10

99
10

Ss OV 0
0

Ss MV2 1
10

100

100
100 0

100

Sla OV1 14
s
100
10

100

Ss aMV 3
99
0

99
100

C O
100
10

A MV 3
99

S RM urV
S
100
100
100

Fp MV 5
0

cle

Ss sOV V 1-1
100
10

Bc V3 1 P O 1
ro

M V
Ss cM V4 3 C c O V1 6
M cO LV 5
u

B pM V
l

O ul 1
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F sM
Ss Ss V3- V2

vir

LV V1
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M MV WX

S
M nM

1
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Ep sVC 7
Ss V7-W 12

us
C sV V
Ss S

S V X

V
W aN
Ss sMV 20

M
Tu N l V 3 1
Ss V3 7

ag
H NlV 36
0
M

W MV
Ss V37

ou
Ss MV1
MV 6

Sn MV4
M

SsM 11
Ss MV

liv
Bc V4
AbMV14
M

SsM V39
SN V1

Ou
SsM V2
CrM lV7

iru
FpM V1
HNlV V1

CfM V38
SsM 25

rm
CcMV 32

SsM
SsMV a

SsM 5

s
15

SsMV 8
7

SsMV
1

SsMV2

BcMV2
SsMV9
SsMV19

CfMV1
V

SsMV33

ia
AbMV1
AaMV1
SsMV1

SsMV8 FpMV1

in

vi
V1

Mi
ru
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Ou bra

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1

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0.5 irid
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Fungi Invertebrates Plants Bacteria Oomycetes Marine microorganisms

Figure 1. Genome organizations of narna-like viruses and phylogenetic relationships of the members in the ‘Narna-Mito-Botourmia’ clade. (A) Genome structure mod-
els for the narna-like viruses identified in this study. The colored boxes indicate hypothetical ORFs. (B) The ML tree was constructed based on the multiple amino acid
sequence alignment of the core conserved domain of RdRps using IQ-TREE with the best-fit model ‘Blosum62þFþR7’. The first identified viruses are marked by red col-
ors, and the identified polynarnaviruses from NCBI SRA or TSA database are marked by blue colors. Sequence information of all selected viruses were supplied in
Supplementary Table S4. The numbers next to each branch represent the bootstrap support based on 1,000 replicates. All branch lengths are drawn to a scale of amino
acid substitutions per site. Host taxa are shown by circles delineated in different colors. The abbreviation ‘WX’ represents Wuxue (WX) city, which was used to distin-
guish the identified viruses in this study from the previously reported mycoviruses with more than 90% amino acid identity.

Botoulivirus, and another three belong to the genus Scleroulivirus
(Fig. 1B).
We identified a novel virus belonging to the family
Fusariviridae, tentatively named Sclerotinia sclerotiorum fusar-
ivirus 2 (SsFV2). The assembled genome of SsFV2 is 6,281 nt in
length and encodes two nonoverlapping ORFs (Supplementary
Fig. S6A). ORF1 encodes a polyprotein with conserved RdRp and
helicase (Hel) domains showing 51.29% identity with previously
described Rutstroemia firma fusarivirus 1. ORF2 encodes a hy-
pothetical protein of unknown function. Phylogenetic analysis
based on the conserved RdRps showed that SsFV2 and other
members of the Fusariviridae family are divided into three
branches with good support (Supplementary Fig. S6B). For this
reason, we propose the division of this family into three genera:
Alpha-, Beta-, and Gammafusarivirus, among which SsFV2
belongs to the Alphafusarivirus genus.
One mycovirus, Sclerotinia sclerotiorum virga-like virus 1
(SsVV1), was identified. The SsVV1 genome consists of two RNA
segments, designated as RNA1 (1,800 nt) and RNA2 (1,878 nt),
which encode RdRp and methyltransferase (Met), respectively
(Supplementary Fig. S7A). The predicted amino acid sequence
of RNA1 showed 44% identity with the RdRp of Agaricus bispo-
rus virus 16 (AbV16; KY357502). The Met encoded by RNA2 of
SsVV1 had an identity of 29% with AbV16. AbV16 is a proposed
member of the Mycovirgaviridae family (Nerva et al. 2019),
whose members exhibit four RNA segments, containing a single
ORF: segment 1 encoding RdRp, segment 2 encoding Met, and
segments 3 and 4 encoding proteins of unknown function
(Deakin et al. 2017). Although both RNA1 and RNA2 of SsVV1
were similar to those of AbV16, we failed to detect the other two
segments corresponding to those of AbV16 from our data.
Phylogenetic analysis revealed that SsVV1 clusters in a clade
with AbV16 along with several other viruses recently discovered
in soil samples and fungal transcriptome data (Supplementary
Fig. S7B).
We also identified three mycoviruses belonging to the
Deltaflexiviridae family, tentatively named Sclerotinia sclerotio-
rum deltaflexivirus 1-WX (SsDFV1-WX), SsDFV2-WX, and
SsDFV3. The replicases of SsDFV1-WX and SsDFV2-WX shared
99% and 98% identity with SsDFV1 (NC_038977) and SsDFV2
(NC_040649), respectively (Supplementary Fig. S8). The assem-
bled genome of SsDFV3 consisted of 7,375 nt with two predicted
ORFs. ORF1 encodes a putative replication-associated polypro-
tein and contains three conserved domains: Met, Hel, and RdRp
(Supplementary Fig. S7A). A BLASTp search using the deduced
amino acid sequence showed that ORF1 has a low identity
(<40%) with the replication-associated polyprotein of members
in the Deltaflexiviridae family (Supplementary Fig. S8).
Phylogenetic analysis revealed that SsDFV3 was in a well-
supported clade with other deltaflexiviruses (Supplementary
Fig. S7B).
A 4470 nt contig was similar to barnavirus, tentatively
named Sclerotinia sclerotiorum barnavirus 1 (SsBNV1). SsBNV1
was a monosegmented þssRNA virus with four ORFs
(Supplementary Fig. S9A). The four ORFs encoded a protein of
unknown function, a putative serine protease domain, RdRp,
and a capsid protein. The predicted RdRp shared 39% identity
with the Mushroom bacilliform virus belonging to the family
Barnaviridae. SsBNV1 within a well-supported clade of the
Barnaviridae family is closely related to members of
Solemoviridae and Luteoviridae families (Supplementary Fig. S9B).
In addition to the mycoviruses mentioned above, other
detected þssRNA viruses and dsRNA viruses included members
of Mitoviridae, Gammaflexiviridae, Tymoviridae, Hypoviridae,
J. Jia et al. | 5
Solemoviridae, Ambiguiviridae, Endornaviridae, Botybirnavirus, and
one unassigned virus species. Due to space limitations, these
mycoviruses are described in Supplementary Text S1.
3.3 Novel negative-sense single-stranded RNA viruses
We identified five -ssRNA mycoviruses belonging to the family
Mymonaviridae. The sequences of these five mycoviruses ranged
from 9,623 to 9,982 nt, suggesting that the complete or nearly
Downloaded from https://academic.oup.com/ve/article/7/1/veab032/6206434 by Universidade Federal de Viçosa user on 25 March 2022
complete genomes of all these viruses were obtained based on
the deep-sequencing data. The L proteins of Sclerotinia sclero-
tiorum negative-stranded RNA virus 1 (SsNSRV1-WX) and
SsNSRV2-WX shared 100% and 99% identity with SsNSRV1
(KJ186782.1) and SsNSRV2 (KP900931.1), respectively
(Supplementary Fig. S10A). Similar to that of other members of
Mymonaviridae, the genome of SsNSRV9 contained five nonover-
lapping ORFs (ORF I–V) arranged linearly in the viral genome
(Supplementary Fig. S10B). The protein encoded by ORF IV
in SsNSRV9 shared 56% identity with the L protein in SsNSRV1
and SsNSRV3 (Supplementary Fig. S10A). In addition, a con-
served gene-junction sequence (30 -UAUU(U/
A)AAUAAAACUUAGGAA-50 ) was found downstream of each
ORF (Supplementary Fig. S10D), which is a common feature in
mononegaviruses (Whelan et al. 2004). Notably, SsNSRV10
and SsNSRV11 contained four ORFs (ORF I–IV) (Supplementary
Fig. S10B), but there was only a transcription start signal with
no stop signal upstream of ORF I and only a transcription
stop signal with no start signal downstream of ORF IV
(Supplementary Fig. S10D); therefore, we believe that these two
mycoviruses may have only four ORFs. However, SsNSRV9 is
similar to the previously reported SsNSRV1 in that there is no
transcription initiation signal in their 3’-UTR (Liu et al. 2014).
Phylogenetic analysis of the L protein confirmed that SsNSRV10
and SsNSRV11 formed an independent evolutionary branch,
while SsNSRV9 was clustered with SsNSRV1 and related viruses
(Fig. 2).
Sclerotinia sclerotiorum ophiovirus-like virus 1 (SsOpLV1)
with 7,817 nt in length shows the highest similarity to Fusarium
poae negative-stranded virus 1 within the recently proposed
Mycoaspiviridae family (Chiapello et al. 2020). SsOpLV1
contained two linearly arranged nonoverlapping ORFs
(Supplementary Fig. S10C). The ORF1 encoded a protein (143 aa)
of unknown function, and ORF2 encoded a polyprotein with the
RdRp domain. Phylogenetic analysis based on RdRp indicated
that mycoophioviruses are closely related to the viruses of the
Aspiviridae family (Fig. 2).
Seven novel mycoviruses related to the members of
Bunyavirales were identified. The genomes for the seven bunya-
viruses (Sclerotinia sclerotiorum bunyavirus 1-7 (SsBYV1-7))
ranged from 6.4 to 8.0 kb, and contain a single ORF
(Supplementary Fig. S11A). The L proteins encoded by the seven
bunyaviruses shared less than 26% identity with those of
known bunyaviruses (Supplementary Table S2). We failed to
find any additional segments related to bunyavirus M or S pro-
teins in the assembled contig database. The alignment of the
conserved motifs of the L proteins encoded by the seven bunya-
viruses and those in the other reported bunyaviruses indicates
that these mycoviruses contain eight conserved motifs
(Supplementary Fig. S12), which represent highly conserved
central regions of RdRps from members of the Bunyavirales order
(Amroun et al. 2017). The conserved endonuclease domain and
motif G (Supplementary Fig. S11B and C) were also identified in
the N-terminal of the L protein encoded by three bunyaviruses
(SsBYV5-7). However, the endonuclease domain and motif G
 6 | Virus Evolution, 2021, Vol. 7, No. 1

100 Sclerotinia sclerotiorum negative-stranded RNA virus 1-WX

100 Sclerotinia sclerotiorum negative-stranded RNA virus 1 | NC_025383
99 Sclerotinia sclerotiorum negative-stranded RNA virus 3 | NC_026732
95 Sclerotinia sclerotiorum negative-stranded RNA virus 9
100 Soybean leaf-associated negative-stranded RNA virus 2 | KT598227
Fusarium graminearum negative-stranded RNA virus 1 | MF276904
100 Soybean leaf-associated negative-stranded RNA virus 1 | KT598225
100 100 Alternaria tenuissima negative-stranded RNA virus 1 | MK584852
100 Sclerotinia sclerotiorum negative-stranded RNA virus 7 | MF444285
100 Botrytis cinerea mymonavirus 1 | MH648611
82 Penicillium cairnsense negative-stranded RNA virus 1 | MK584851
Sclerotinia sclerotiorum negative-stranded RNA virus 2-WX Mymonaviridae
100
100 Sclerotinia sclerotiorum negative-stranded RNA virus 2 | KP900931
87 100 Sclerotinia sclerotiorum negative-stranded RNA virus 4 | NC_043483

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Soybean leaf-associated negative-stranded RNA virus 3 | KT598228
100 Sclerotinia sclerotiorum negative-stranded RNA virus 11
Sclerotinia sclerotiorum negative-stranded RNA virus 10
100 Soybean leaf-associated negative-stranded RNA virus 4 | KT598229
Hubei rhabdo-like virus 4 | KX884403
Lentinula edodes negative-strand RNA virus 1 | LC466007
93
94 100 Penicillium glabrum negative-stranded RNA virus 1 | MK584853
Penicillium adametzioides negative-stranded
g RNA virus 1 | MK584858
100 Rhabdoviridae
Lishi Spider Virus 2 | NC_031259 Lispiviridae
p
100 Paramyxoviridae
y
99
98
94 100 Filoviridae
100
Pneumoviridae
100 Nyamiviridae
y
100
Bornaviridae
84 Xi h
Xincheng M
Mosquito
i Vi
Virus | NC
NC_031244
031244 Xinmoviridae
Pteromalus
Pt l puparum negative-strand
ti t d RNA virusi 1 | NC_038269
NC 038269 Artoviridae
100 99 Rhi
Rhizoctonia
t i solani
l i negative-stranded
ti t d d virus
i 2 | KP900920
100 Rhizoctonia solani negative-stranded virus 3 | KP900903
100 Rhizoctonia solani negative-stranded virus 1 | KP900919 Mycoaspiviridae
100 Cladosporium cladosporioides negative-stranded RNA virus 1 | MK584856
100 100 Fusarium poae negative-stranded virus 1 | NC_030871
100
Sclerotinia sclerotiorum ophiovirus like virus 1
Aspiviridae
100
98
100 Sclerotinia sclerotiorum negative-stranded RNA virus 5 | KF913892
Alternaria tenuissima negative-stranded RNA virus 2 | MK584855
100
100 Laurel Lake virus | KX774630 Laulavirus Phenuiviridae
100 Citrus concave gum-associated virus | NC_035759 Coguvirus
100 Lentinula edodes negative-strand RNA virus 2 | LC466008
Entoleuca phenui-like virus 1 | MF375882
100 100 Sclerotinia sclerotiorum bunyavirus 5
100 Sclerotinia sclerotiorum bunyavirus 6
99 Sclerotinia sclerotiorum bunyavirus 7 proposed
93 Wuhan Spider Virus | KM817699 Mycophenuiviridae
99 Fusarium poae negative-stranded virus 2 | NC_030872
88 99 100 Penicillium roseopurpureum negative ssRNA virus 1 | MG887749
Coniothyrium
y diplodiella
p negative-stranded
g RNA virus 1 | MK584854
Leptomonas
p moramango g leishbunyavirus
y 1 | KX280012 Leishbuviridae
100
99 100 Arenaviridae
100 Hubei myriapoda
y p virus 5 | NC_033761
_ Mypoviridae
yp
100
100 Nairoviridae
90 Wuhan Millipede
p Virus 2 | KM817696 Wupedeviridae
p
100 Rhizoctonia solani negative-stranded virus 4 | KP900923
Cladosporium cladosporioides negative-stranded
g RNA virus 2 | MK584857 New group?
100 Macrophomina phaseolina negative-stranded RNA virus 1 | KP900899
Botrytis
y cinerea negative-stranded
g RNA virus 1 | NC_028466
_ New group?
100 100
97 Peribunyaviridae
y
100 98 European mountain ash ringspot-associated emaravirus | NC_013105 Fimoviridae
W li crustacean
Wenling t virus
i 9 | NC
NC_032143
032143 Cu
Cruliviridae
dae
94 Tomato spotted
T d wilt
il tospovirus
i | NC
NC_002052
002052
100
Tospoviridae
p
100
Hantaviridae
89 Phasmaviridae
100 Sclerotinia sclerotiorum bunyavirus 1 proposed
87
100 Sclerotinia sclerotiorum bunyavirus 2
100 Sclerotinia sclerotiorum bunyavirus 3 Sclerobunyaviridae
Sclerotinia sclerotiorum bunyavirus 4
Fungi Plants Vertebrates Invertebrates
0.5
Vertebrates and Invertebrates Vertebrates, Invertebrates and plants

Figure 2. ML phylogenetic analysis depicting the evolutionary relationships of the negative-stranded RNA viruses. The best-fit model for constructing the phylogenetic
tree is ‘LGþFþR8’. The name of the virus family or genus is shown on the right. This is same as that of Fig. 1.

were not detected in the L proteins encoded by the four other We therefore propose the establishment of a new family,
bunyaviruses. A phylogenetic tree was constructed using the Mycophenuiviridae, to accommodate these similar bunyavi-
core regions of the L protein. Seven sclerotinia bunyaviruses ruses that infect fungi and invertebrates (Fig. 2).
were assigned into two clades. Four bunyaviruses (SsNYV1-4)
were well supported in one phylogenetic cluster, which was sig-
3.4 Diversity of the S. sclerotiorum virome in a single
nificantly distant from the other bunyaviruses, suggesting that
rapeseed field
these four bunyaviruses represent a new virus lineage.
Therefore, we propose the establishment of a new family, We examined the proportion of mycovirus-associated reads in
Sclerobunyaviridae, to accommodate these similar bunyavi- each library, which we found to be 4.07% for 2016SS, 9.51% for
ruses. The other three bunyaviruses (SsNYV5-7) formed well- 2017SS, and 2.89% for 2018SS (Fig. 3A). The sixty-eight mycovi-
supported clades with an invertebrate virus (Wuhan spider vi- ruses we identified from these three libraries represent four ge-
rus) and several fungal bunyaviruses but were significantly phy- nome types: positive-sense single-stranded RNA (þssRNA),
logenetically distant from members of the Phenuiviridae family. negative-sense single-stranded RNA (-ssRNA), double-stranded
 J. Jia et al. | 7

A D
25 Genome type
2018
5 +ssRNA
2018 4
1 −ssRNA
2017
dsRNA
33 ssDNA
2016 10
2017 8
1
0

10

20

30

40

50

60

70

80

90

100
Proportion 36
2016 6
S. sclerotiorum Viruses Others 4

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1
B ssDNA,1,1.5% 0 5 10 15 20 25 30 35 40
Number of viruses
dsRNA,11,16.2% E
Year Virus Genus Virus Family Genome Type
Fusariviridae
Fusariv
vviridae
Penoulivirus
Alphafusariviruses
Alphafu
p usarivirusviruses
ruse
+ssRNA,43,63.2%
Botouliivirus
2016 Hbsclerovirus
Hb
Hbscle
lerovirus
i Botourm
rmiaviridae
rm
Unassigned
Unassi igned genus
gen
genu
nus
−ssRNA,13,19.1%
Sclerouulivirus Solemoviridae
Solemo
oviridae
o
Unassigned fam
Unassigned family
famii y
Betaen
ndornavirus Endorn
naviridae
n +ssRNA
Gammahypoviruses
Hypoviiiridae
Unclassified,1,2.3%
Solemoviridae,1,2.3% Mitovirrus
C Barnaviridae,1,2.3% Mitoviri
riidae
Fusariviridae,1,2.3% Betahy
Betahypoviruses
ypoviruse es
Mycovirgaviridae,1,2.3% Deltafle
exivirus Deltafle exiviridae
e aee
Botourmiaviridae,12,27.9% Tymoviridae,1,2.3% 2017 Fusagravirus
Fusagr
gravirus Ambiguiviridae
A mbigu uiviridae
u
Gammaflexiviridae,1,2.3% Mycotombusvirus
Mycoto
y ombusvirus svirus
virus Mycovirgaviridae
Mycovirgavirid
M
Mycovi
y iirgaviridae
rgaviridae
Mycovirgavirus
M
Mycov i
irgavirus
i Gammaflexiviridae
Gamm
G aflexiviri
fl i i id
daae
ae
Ambiguiviridae,2,4.7% Mycoflexivirus
Mycofle
M flexivirus
i i Fusagraviridae
Fusagr rraviridae
dae
Barnavirus
Barnav
B virus
i Barnaviridae
Barnav
B vviridae
i id e
Polynarnaviridae,2,4.7% Mycotymovirus
Mycoty
y ymovirus Tymoviridae
Tymovi
y iiridae
Narnaviridae
Narnav vviridae
dsRNA
Mycobunyavius
Mycob unyavius
Narnaviridae,2,4.7% Narnavirus
Narnav virus Botybirrrnaviridae
e
Botybirrnavirus
Mycobu
y unyaviridae
u yavirida
Deltaflexiviridae,3,7% Mycophenuivirus
Mycophenuivirus
us Polynarnaviridae
Polyna rnaviridae -ss
ss
ssRNA
Mitoviridae,7,16.3% Polyna
y arnavirus Mycophenuivirid
Mycophhenuiviridae
h ridae
dae
Endornaviridae,4,9.3% 2018
Sclerottimonavirus Mymon
naviridae
n
Hypoviridae,4,9.3% G ycircularvirus
Gemycircularvirus
Gemyc i l i Genom
Genomoviridae
moviridae
m e
Mycoaspivirus
Mycoa spivirus Mycoaspiviridae
Mycoas
sspivirida ssDNA

Figure 3. The diversity of mycoviruses detected in S. sclerotiorum. (A) Proportion of each taxonomic category in each library based on read counts. (B) The percentage
of each virus genome type. (C) The percentage of each positive, single-stranded RNA virus at the family level. (D) Mycoviruses belonging to different genome types
presented in each library. (E) Sankey diagram showing the compositions of mycovirome from different years of S. sclerotiorum field isolates.

RNA (dsRNA), and single-stranded DNA (ssDNA) (Fig. 3B).
Among these sixty-eight mycoviruses, the amino acid sequen-
ces of twenty-two mycoviruses showed high identity (90%) to
previously reported viruses, twenty-eight mycoviruses repre-
sented novel mycoviruses sharing low amino acid sequence
identity (<70%) with previously recognized viruses, and eigh-
teen mycoviruses had 70% and <90% amino acid identity with
previously recognized viruses (Supplementary Table S2). The
characteristics of all identified mycoviruses, including their
contig length and best hit in the NCBI-nr database, are summa-
rized in Supplementary Table S2. Based on the amino acid
sequences of the replicase encoded by the assembled contigs,
all identified mycoviruses were classified. Nineteen of sixty-
eight (27.9%) mycoviruses had a lower identity (<45%) than pre-
viously characterized viruses, and eleven of these nineteen
could not be placed into any established family. At the genus
level, thirty-eight mycoviruses could be assigned to eleven
established genera, while the remaining thirty mycoviruses
could be assigned to thirteen proposed genera and one unas-
signed genus.
þssRNA mycoviruses were the most diverse class within the
three libraries, compared to other types (Fig. 3B). Forty-three
mycoviruses were þssRNA viruses, accounting for up to 63.2%
of the identified viruses, suggesting that S. sclerotiorum
harbors abundant þssRNA mycoviruses with high diversity
(Fig. 3B). Among the þssRNA mycoviruses, members of the
Botourmiaviridae and Mitoviridae families were most prevalent,
accounting for up to 44.2% (Fig. 3C). The eleven identified
dsRNA mycoviruses included seven botybirnaviruses and four
fusagraviruses (Supplementary Fig. S13). We also identified an
ssDNA mycovirus that shared 100% identity to Sclerotinia scle-
rotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) be-
longing to the Genomoviridae family. Finally, the mycoviruses
with -ssRNA genomes were classified into three orders:
Bunyavirales (seven viruses), Mononegavirales (five viruses), and
Serpentovirales (one virus). These results suggest that mycovi-
ruses present in S. sclerotiorum are diverse, even though all iso-
lates originated from a single rapeseed field.
3.5 Interannual dynamics of members in the
S. sclerotiorum virome
The composition of virome was different among the three
libraries. The list of putative mycoviruses was summarized for
each library, and we found that 47, 52, and 35 different mycovi-
ruses were detected in the 2016SS, 2017SS, and 2018SS libraries,
respectively (Fig. 3D). In all three libraries, the number of
þssRNA mycoviruses was greater than that of -ssRNA mycovi-
ruses. The number of dsRNA mycoviruses was less than that of
-ssRNA mycoviruses, and the number of ssDNA mycoviruses
was the lowest, with only one of these viruses found in all
 8 | Virus Evolution, 2021, Vol. 7, No. 1

libraries (Fig. 3D). The libraries and the taxonomy of mycovi-
ruses were summarized in a Sankey diagram (Fig. 3E).
Mycovirus occurrence changed within sampling times as fol-
lows: 35.3% of mycoviruses were detected in all three years,
25.8% were detected twice, and 39.4% were detected once. More
than 60% of the identified dsRNA mycoviruses and -ssRNA
mycoviruses were detected once, suggesting that these mycovi-
ruses may have difficulty spreading widely in S. sclerotiorum or
that they may be easily eliminated. In total, twenty-four myco-
the Botybirnavirus genus, and one ssDNA mycovirus in the
Gemycircularvirus genus (Supplementary Table S2).
The relative abundances of the identified viral families
showed differences among the years. Mycoviruses within the
Mitoviridae family were the most abundant, and their TPM
values accounted for more than 63.4% of all mycovirus TPM val-
ues (Fig. 4B). Botourmiaviruses were the second most common
after mitoviruses in the 2016SS library, accounting for 23.2% of
the mycovirus TPM values, while the family accounted for only

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viruses were found each year, accounting for 35.3% of all identi-
fied viruses (Fig. 4A), suggesting that these mycoviruses
commonly exist in the population of S. sclerotiorum and are po-
tential core mycoviruses in the virome of S. sclerotiorum. Among
these twenty-four mycoviruses, nineteen were þssRNA mycovi-
ruses, including five mitoviruses, five botourmiaviruses, three
hypoviruses, two deltaflexiviruses, a narnaviruses, a gamma-
flexivirus, a tymovirus, and a betaendornavirus. Additionally,
there are five other mycoviruses, including three -ssRNA myco-
viruses in the Sclerotimonavirus genus, one dsRNA mycovirus in
2.0% in the 2018SS library. In the 2017SS library, the abundance
levels of mycoviruses within the Botybirnavirus genus and
Botourmiaviridae family were lower than the abundance of
mycoviruses belonging to the Mitoviridae family, accounting for
12.4% and 6.8% of the TPM values, respectively. In the 2018SS li-
brary, the abundance of mycoviruses within the Mymonaviridae
family accounted for more than 17.3% of the total abundance,
while mycoviruses in this family accounted for less than 2.0% in
the other two libraries. A heat map of the relative abundances
of all mycoviruses identified in the different libraries is pre-
sented in Fig. 4C. As shown in the heat map, almost all the

A C 100 SsBV1
2016 2017 Not detected 98 SsBV2 Sclerobunyaviridae
SsBV3
1.2 100 SsBV4
98 SsBV5
0.9 SsBV6 Mycophenuiviridae
100 SsBV7
9 0.6 76 SsNSRV11
9 15 95 SsNSRV10
0.3 SsNSRV2-WX Mymonaviridae
SsNSRV1-WX
0.0 95 99 SsNSRV9
24 SsOpLV1 Mycoaspiviridae
-0.3 92 SsGFV1 Gammaflexiviridae
5 4 92 SsMTV1-WX Tymoviridae
-0.6 SsULV3-WX1 Ambiguiviridae
71 98 100 SsULV3-WX2
-0.9 94 SsDFV1-WX
SsDFV3 Deltaflexiviridae
2 -1.2 100 SsDFV2-WX
99 SsEV8
86 100 Endornaviridae
SsEV1-WX
80 SsEV9
SsBNV1 Barnaviridae
2018 88 HuSRV1-WX Solemoviridae
85
SsVV1 Mycovirgaviridae
B 94 SsBRV4
100 Family/Genus 99 SsBRV1-WX
SsBRV5-WX1 Botybirnavirus
100 96 SsBRV5-WX2
Mycoaspiviridae 84 SsBRV3-WX
Mycovirgaviridae SsBRV2-WX
98 93 SsFGV1
Narnaviridae SsFGV2 Fusagraviridae
Solemoviridae 100 SsNsV-L-WX1
75 Fusariviridae SsNsV-L-WX2
97 SsHV8-WX1
Relative abundance (%)

Unclassified 99 SsHV8-WX2 Hypoviridae

Tymoviridae SsHV2-WX
99 SsHV7
Mycophenuiviridae 97 SsFV2 Fusariviridae
Endornaviridae SsOV10
100 SsOV11
Gammaflexiviridae SsOV12
50 Sclerobunyaviridae 100 SsOV13
93 SsOV5
Deltaflexiviridae SsOV6 Botourmiaviridae
97 100 SsOV7
Barnaviridae 98 SsOV15
Fusagraviridae 99 SsOV16
Hypoviridae 89 SsOV14
100 SsOV8
Genomoviridae SsOV9
25 100 SsNV1 Polynarnaviridae
Mymonaviridae
SsNV2
Botybirnavirus 99 SsNV3 Narnaviridae
Polynarnaviridae 70 100 SsNV4
100 SsMV37
Ambiguiviridae 91 SsMV3-WX
94
Botourmiaviridae SsMV35 Mitoviridae
SsMV7-WX
0 Mitoviridae 99 100 SsMV36
2016 2017 2018 SsMV39
100 SsMV38
SsHADV1-WX Genomoviridae
2016 2017 2018

Figure 4. The abundances of mycoviruses in S. sclerotiorum from different years. (A) The number of shared mycoviruses in the virome of S. sclerotiorum in three years. (B)
The relative abundance of mycoviruses at the family level in the same year, normalized by column. (C) Heat map displaying the relative abundance of the mycoviruses
in different years according to the TPM values, normalized by row. The red columns show high abundance, and grey columns indicate not detected. Mycoviruses
detected in all three libraries are marked with green dots.

mycoviruses had changing abundances between sampling
years.
4. Discussion
Metatranscriptomics sequencing has dramatically expanded
our understanding of virus diversity and allowed novel mycovi-
ruses to be increasingly discovered in phytopathogenic fungi
(Marzano et al. 2016; Kotta-Loizou and Coutts 2017; Mu et al.
2017; Arjona-Lopez et al. 2018; Nerva et al. 2019; Chiapello et al.
2020). However, the understanding on the interannual dynam-
ics of mycovirus under natural conditions is limited at a popula-
tion level. In the present research, we performed viral
metatranscriptomics sequencing to investigate the interannual
dynamics and characterize unbiased viral communities in S. scle-
rotiorum collected from a single rapeseed-growing field for three
consecutive years. As expected, the species and number of the
identified mycoviruses showed variation in an individual year,
but it is noteworthy that twenty-four mycoviruses detected in all
three years are considered to be core mycoviruses in S. sclerotio-
rum virome. Additionally, some of the mycoviruses discovered in
this study were classified into families of Narnaviridae,
Polynarnaviridae, Gammaflexiviridae, Mycovirgaviridae, Barnaviridae,
Mycoaspiviridae, Mycophenuiviridae, Sclerobunyaviridae with
S. sclerotiorum as the host for the first time. The findings enrich
our understanding on the diversity of mycoviruses and provided
important evolutionary clues.
In this study, sixty-eight mycoviruses were detected in
S. sclerotiorum, and besides one unclassified mycovirus, the
other sixty-seven mycoviruses were assigned into twenty fami-
lies, suggesting that S. sclerotiorum harbors a highly complex
mycovirus composition, even in a single crop field. Moreover,
twenty-eight mycoviruses were previously uncharacterized, in-
dicating mycoviral communities in S. sclerotiorum are still poorly
covered by current databases. Two virome analyses were previ-
ously conducted in S. sclerotinia. Twenty-eight mycoviruses
were detected from 138 isolates of S. sclerotiorum in the USA, be-
longing to eight families (nine genera) (Marzano et al. 2016), of
which members from seven families (eight genera) were found
in the present research. Fifty-seven mycoviruses were detected
from eighty-four isolates in Australia, belonging to thirteen
families (fourteen genera) (Mu et al. 2017), of which members
within ten families (eleven genera) were detected in our sam-
ples. Mitoviruses are the most prevalent in S. sclerotiorum sam-
ples from Australia and the USA, and the proportion of them in
those two viromes of S. sclerotiorum are 43.9% (25/57) and 60.7%
(17/28), respectively. Unlike samples from the United States and
Australia, botourmiaviruses were most prevalent in this virome
survey of S. sclerotiorum from China, accounting for 17.6% (12/
68), followed by mitoviruses (7/68) and botybirnaviruses (7/68).
Based on the percentage of genome types, our findings were
similar to those of previously reported results (Marzano et al.
2016; Mu et al. 2017), with þssRNA viruses as the dominant pop-
ulation accounting for more than 60% of the identified mycovi-
ruses. S. sclerotiorum virome showed a similar mycovirus
composition despite location as S. sclerotiorum isolates from
Australia, USA, and China are from different continents sepa-
rated by oceans. Therefore, we boldly speculated that the inter-
action system between S. sclerotiorum and its core virome had
been initiated before continental plate separation.
The S. sclerotiorum isolates of the USA and Australia were col-
lected from different geographical regions and plant hosts,
while strains of the Chinese group were isolated from a smaller
annual rapeseed-growing field. However, as mentioned above,
J. Jia et al. | 9
the mycovirus composition in a single field was found similar
to the previously reported virome, although the numbers of spe-
cies were more diverse. Moreover, most of these mycoviruses
did not exhibit any specific association to geography or plant
host. This result also revealed that the virome of S. sclerotiorum
from a single crop field in the same region might not differ sig-
nificantly from the overall virome of S. sclerotiorum sampled
from other plant hosts and geographical origins. Whether
viromes in other species of fungal populations have similar
Downloaded from https://academic.oup.com/ve/article/7/1/veab032/6206434 by Universidade Federal de Viçosa user on 25 March 2022
results is still unclear and yet to be explored for the dynamics
among the plant disease severity, fungal hosts, and mycovi-
ruses at population levels.
Previous reports brought insights into the important roles
viruses play in natural ecosystems (Suttle 2007; Dell’Anno et al.
2015). Understanding the temporal dynamics of viromes could
help us determine the drivers of community stability and eco-
system function generally in nature (Rodriguez-Brito et al. 2010;
Arkhipova et al. 2018; Ignacio-Espinoza et al. 2019) and specifi-
cally in agricultural fields. For instance, the virus population
can co-vary with prokaryotic abundance by preying on the
microbes and change the microbial community composition,
and thereby modulating ecosystem functions such as carbon
and nitrogen cycling in the natural environment (Suttle 2007).
The temporal dynamics of several environmental viromes have
been characterized in recent decades, including bioaerosol
(Prussin et al. 2019), ocean (Chow and Fuhrman 2012), sphag-
num peatlands (Ballaud et al. 2016), and sediment ecosystems
(Helton et al. 2012). In this study, we found that the mycovirus
communities exhibited fluctuating temporal abundance, with
the viral populations in 2017 being larger than those in the other
two years. In contrast to mycoviruses, another study analyzed
plant viruses from six native plant species over four years in the
Nature Conservancy’s Tallgrass Prairie Preserve, Osage County,
Oklahoma, USA. The taxonomic composition of plant viruses
was found to have a close relationship with the plant host spe-
cies but no clear relationship with the sampling site or year
(Thapa et al. 2015). Although most mycoviruses cause latent
infections, the mycoviruses associated with hypovirulence/
hypervirulence or other biological functions have been widely
reported in various phytopathogenic or nonpathogenic fungi
(Marquez et al. 2007; Ghabrial et al. 2015; Kotta-Loizou and
Coutts 2017; Nerva et al. 2019). Mycoviruses also can influence
the plant–endophyte relationship by changing the virulence or
fitness of the fungal host under altered environmental condi-
tions (Bao and Roossinck 2013). Bio-priming treatment of rape-
seeds with the hypovirulent strain DT-8 infected with a
mycovirus, SsHADV-1, could influence the structure and com-
position of overall fungal communities in Sclerotinia stem rot
(Qu et al. 2020). Therefore, we believe that mycoviruses may
play an essential role in fungal communities in the agricultural
ecosystem via complex fungi–mycovirus interactions. However,
the temporal dynamics of virome have not been explored at a
fungal community level. Therefore, our study represents the
first effort in quantitatively and qualitatively describing the
temporal dynamics of the virome in fungi. Previous research
confirmed that the occurrence of sclerotinia disease was
affected by many factors including temperature, humidity,
and host resistance (Sharma et al. 2015). However, beyond
abiotic and host factors, we speculated that the virulence of the
S. sclerotiorum population taken as a whole was also greatly
affected by mycoviruses for the following reasons. First, the
isolates of S. sclerotiorum collected from a single crop field
showed significant virulence variation (Attanayake et al. 2013;
Abán et al. 2018), and a wide variety of mycoviruses have been
 10 | Virus Evolution, 2021, Vol. 7, No. 1
discovered in S. sclerotiorum (Jiang et al. 2013; Marzano et al.
2016; Mu et al. 2017). Secondly, some mycoviruses could not
only confer hypovirulence on S. sclerotiorum but also have the
potential to convert S. sclerotiorum into an endophyte and en-
hance the resistance of rapeseed against the phytopathogens,
for example Botrytis cinerea and S. sclerotiorum (Zhang et al. 2020).
Third, biopriming with hypovirulent strain DT-8 could reduce
the total abundance of the potential phytopathogenic fungi (Qu
et al. 2020). Therefore, we speculate that the interannual dy-
namics of virome in S. sclerotiorum may be the hidden driver in
shaping the fungal community structure that then may reduce
the occurrence of diseases caused by other fungal pathogens.
It is generally thought that the RdRp of all RNA viruses is
encoded by a single segment of gene and the phylogeny of RNA
viruses is fully characterized based on the viral RdRp
(te Velthuis 2014; Wolf et al. 2018). We found the RdRps of two
novel multi-segmented polynarnaviruses encoded by two dif-
ferent segments and this similar result was recently reported in
Aspergillus fumigatus, Magnaporthe oryzae, and Oidiodendron maius
(Chiba et al. 2020; Sutela et al. 2020). By analyzing NCBI SRA and
TSA data, we found that seventeen polynarnaviruses containing
multi-segmented genomes with their RdRp conserved domains
located in different segments. This finding suggests that multi-
segmented polynarnaviruses are widely distributed in fungi,
and their genomes possibly contain up to seven segments.
Based on the understanding of the lifestyle of multipartite vi-
ruses and multi-segmented viruses (Lowen 2018), the genome
segmentation of polynarnaviruses may be advantageous for vi-
ral replication and recombination. We also found two narnavi-
ruses, SsNV3 and SsNV4, with bi-segmented genomes. Based on
Phyre2 results, the protein encoded by RNA 2 of SsNV3 showed
the similarity to a -ssRNA virus, but I-TASSER results indicated
the similarity to dsRNA viruses. We speculated that the differ-
ence between the two software results could be due to the poor
conservation of viral coat proteins, and the 3D structure of most
viral coat proteins has not been resolved, and there is no avail-
able adequate data related to the narnavirus coat proteins in
the database. This may also be the reason why SsNV4 did not
predict the information associated with the viral coat protein.
We believed that more narnaviruses that could have coat pro-
teins would be identified with the release of these two viral
sequences in the future. We speculated that these two narnavi-
ruses may have obtained genes related to coat proteins from
other viruses or hosts, or that capsidless narnaviruses have lost
their coat protein genes during the evolutionary process. These
two viruses revealed an unanticipated evolutionary link be-
tween encapsidated and capsidless RNA viruses. In summary,
these newly characterized mycoviruses with unique genome
features have a distant phylogenetic relationship from all
known viruses and formed an independent evolutionary lineage
that supported the establishment of a new genus or family.
Acknowledgments
We would like to thank Mr. Huang Huang in Huazhong
Agricultural University and Mr. Zaiwu Zheng in the Plant
Protection Station of Wuxue City of Hubei Province for his
help in collecting samples, Dr. Kenichi Tsuda from
Huazhong Agricultural University for constructive sugges-
tions, and Amanda Gutek for proof-reading the manuscript.
This work was financially supported by the National Science
Foundation of China (31722046), the National Key Research
and Development Program of China (2017YFD0201100), and
the earmarked fund for China Agriculture Research System
(CARS-12).
Supplementary data
Supplementary data are available at Virus Evolution online.
Conflict of interest: None declared.
Downloaded from https://academic.oup.com/ve/article/7/1/veab032/6206434 by Universidade Federal de Viçosa user on 25 March 2022
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